Cell survival and arsenic carcinogenesis

描述（由申请人提供）：虽然砷致癌的分子机制仍有待研究，但砷产生的活性氧（ROS）被认为是重要的。砷生成的ROS可引起DNA损伤、脂质过氧化和蛋白质修饰，导致各种致癌反应。我们的初步研究表明，砷转化的人肺支气管上皮细胞（BEAS-2B）相对于未转化的细胞，砷诱导的ROS生成能力大大降低。这种ROS生成的减少使细胞具有癌前特征，包括快速生长。砷转化细胞在砷暴露后表现出细胞凋亡减少（细胞凋亡抵抗）。本研究推测，由于ROS生成能力较低，砷转化细胞产生凋亡抵抗，细胞存活率提高，这可能是砷诱导癌变的整体机制。为了验证这一假设，提出了三个目标。目的1将探讨砷转移细胞中ROS生成减少的机制。我们将研究在砷转化的BEAS-2B细胞中，ROS生成途径的损伤是否与砷诱导的低水平ROS生成有关。我们还将研究转化细胞中抗氧化酶水平的增加是否会导致ROS生成水平的降低。这一目的的完成将建立砷转化细胞中ROS生成减少的机制。目的2将探讨减少ROS生成在砷转化细胞凋亡抵抗中的作用。我们研究(a)砷转化细胞的ROS生成减少是否与砷转化细胞的凋亡抵抗有关；(b)砷转化细胞的SOD和过氧化氢酶的抗氧化活性是否高于与其传代匹配的对照细胞；(c)在砷刺激下，转化细胞中抗氧化酶的敲除或NADPH氧化酶的过表达是否会增加ROS的产生，并促进细胞凋亡；(d)敲低Bcl-2是否增加砷转化细胞的凋亡；(e)砷转化后的细胞是否会因ROS生成减少和细胞凋亡抵抗而表现出快速生长和增强侵袭迁移能力。总之，这一目的将证明活性氧在砷转化细胞的凋亡抵抗中的作用。目的3将探讨砷转化细胞诱导肿瘤发生及其凋亡的作用。我们将探讨凋亡抵抗在砷转化细胞肿瘤发生中的作用。我们还将利用皮肤肿瘤模型和原位肺癌模型研究ROS和凋亡调节蛋白Bcl-2在砷转化细胞诱导的肿瘤发生中的作用。预计下调Bcl-2可增加砷转化细胞的凋亡，从而抑制肿瘤生长。同样，细胞产生ROS能力的改变，即通过异位过表达和敲低活性氧清除和产生酶，将对砷转化细胞的肿瘤发生产生影响。

DESCRIPTION (provided by applicant): Although the molecular mechanism of arsenic-induced carcinogenesis remains to be investigated, reactive oxygen species (ROS) generated by arsenic are considered to be important. Arsenic-generated ROS could cause DNA damage, lipid peroxidation and protein modification, leading to various carcinogenic responses. Our preliminary studies have shown that the capacity of ROS generation induced by arsenic is substantially reduced in arsenic-transformed human lung bronchial epithelial (BEAS-2B) cells relative to the non-transformed cells. Such a reduction in ROS generation endows cells with premalignant features, including rapid growth. Arsenic- transformed cells exhibit a decreased apoptosis (apoptosis resistance) upon arsenic exposure. This proposal hypothesizes that due to a low potency of ROS generation, arsenic-transformed cells develop apoptosis resistance and increased cell survival, contributing to the overall mechanism of arsenic-induced carcinogenesis. Three aims are proposed to test this hypothesis. Aim 1 will investigate the mechanism of decreased ROS generation in the arsenic-transferred cells. We will investigate whether impairment of the ROS generating pathway is responsible for a low level of arsenic-induced ROS generation in arsenic transformed BEAS-2B cells. We will also investigate whether the increased level of antioxidant enzymes in transformed cells contributes to the decreased level of ROS generation. The completion of this aim will establish the mechanism of decreased ROS generation in arsenic-transformed cells. Aim 2 will investigate the role of reduced ROS generation in apoptosis resistance of arsenic-transformed cells. We investigate (a) whether reduced ROS generation of arsenic- transformed cells is responsible for apoptosis resistance in arsenic-transformed cells; (b) whether arsenic- transformed cells have higher antioxidant activities of SOD and catalase than their passage-matched control cells; (c) whether knock-down of antioxidant enzymes or overexpressing NADPH oxidase in the transformed cells increases ROS generation and enhances apoptosis in response to arsenic stimulation; (d) whether knocking down of Bcl-2 increases apoptosis of arsenic-transformed cells; and (e) whether arsenic- transformed cell will show fast growth and enhanced invasion and migration due to the decreased ROS generation and apoptosis resistance. Overall, this aim will demonstrate the role of ROS in apoptosis resistance of arsenic-transformed cells. Aim 3 will investigate the arsenic-transformed cells-induced tumorigenesis and the role of apoptosis. We will investigate the role of apoptosis resistance in tumorigenesis of arsenic-transformed cells. We will also investigate the roles of ROS and apoptosis regulatory proteins, Bcl-2, in arsenic-transformed cells-induced tumorigenesis using both skin tumorigenesis model and orthotopic lung cancer model. It is expected that the increase of apoptosis of arsenic-transformed cells by Bcl-2 knockdown will decrease tumor growth. Similarly, alterations in the ROS generating capacity of the cells, i.e., by ectopic overexpression and knockdown of the ROS-scavenging and producing enzymes, will have an effect on tumorigenesis of arsenic-transformed cells.